Method for purifying enzymes

ABSTRACT

INHIBITOR-FREE READILY WATER-SOLUBLE ENZYMES ARE PREPARED BY COMMINUTING ANIMAL PANCREAS WHICH HAS BEEN FROZEN IMMEDIATELY AFTER SLAUGHTERING, EXTRACTING SAID PANCREAS WITH AN ALCOHOL CONTAINING 1-2 CARBON ATOMS, TREATING SAID EXTRACT WITH A WATER-INSOLUBLE LIQUID SOLVENT SELECTED FROM THE GROUP CONSISTING OF A BUTYL ALCOHOL AND AN ETHER, PRECIPITATING SAID ENZYME FROM THE RESULTING CLEAR LAYER WITH A WATER-SOLUBLE SOLVENT SELECTED FROM THE GROUP CONSISTING OF AN ALCOHOL CONTAINING 1-2 CARBON ATOMS AND A KETONE AT A TEMPERATURE BELOW 10*C. AND RECOVERING THE ENZYME.

United States Patent Office 3,691,015 Patented Sept. 12, 1972 3,691,015 METHOD FOR PURIFYING ENZYMES Fritz Leidholdt, Kleinmachnow, Germany, assignor to Colgate-Palmolive Company, New York, NY. No Drawing. Filed Mar. 18, 1970, Ser. No. 20,838 Int. Cl. C07g 7/026 US. Cl. 195--66 R 7 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a method for preparing inhibitor-free, easily water-soluble enzymes which are especially suitable for the use in detergent compositions, as well as to detergent compositions containing same and to a process for making such detergent compositions.

It is known to use powdery or granular detergent compositions containing enzymes. Generally preferred enzymes for this purpose are hydrolases or proteinases, trypsin, esterases, carbohydrases, lipases, and nucleases. In some cases, also oxidases, transferases and transphosphorylases are used alone or in combination. These enzymes can be prepared from plant or animal products or can partly be cultivated bacterially. The use of enzymes in detergent compositions creates substantial difiiculties since the enzymatic soil removal from textile fibers takes place under conditions which are completely diiferent from the analogous effect of single enzymes in vivo. This difference is due to the fact that isolated enzymes behave completely different in vivo in the presence of surface active agents or tensides, at modified pH values or at other temperatures and in the presence of other materials containing in detergent compositions. Thus, e.g. already during isolation of the enzymes, low molecular proteinaceous materials are separated which contain the co-enzymes or co-factors necessary for the activation and which further possess a certain effect as protective colloid. Also, the dry detergent compositions such as washing, cleaning or soaking products and the washing lye contain substances having a detrimental effect on the high-molecular proteinaceous products of which the enzymes are composed; these substances include e.g. the alkali metal salts contained in washing products and effecting an increase of the pH, entrained heavy metal salts, certain tensides contained in washing products, in particular anionic detergents, as well as certain optical brighteners and perfume components.

Other washing products contain sodium perborate as bleaching agent in amounts of e.g. 10 to 20% by weight which liberates active oxygen at temperatures above 60 C. This results in a strong alkalinity of the washing solution whereby non-adapted enzymes of animal, bacterial or plant origin are modified by oxidation and adsorption so that the enzymatic effect is strongly reduced.

Although detergent compositions containing bacterial proteinases are known which are stable in the washing lye up to temperatures of about 80 C. and are hydrolytically active in the presence of sodium perborate even up to a pH of to 10, it has been found that these detergent compositions have only a minor enzymatic effect and that the observed effect is obtained merely due to the bleaching activity of the perborate. Moreover, the magnesium silicates added as stabilizer for sodium perborate as well as all other metasilicates and trisilicates in general have an influence on any proteinase, independent from Whether it is of animal, plant or bacterial origin. In this respect, purified or crystallized proteinases are even less stable than unpurified enzymes since the latter contain colloidal by-products which show a certain protective effect, at least for a short time.

The proteinases prepared by the known methods contain substantial amounts of proteinase inhibitors such as the soy bean inhibitor or the inhibitor of the pancreas of all mammals. Attempts have been made to reduce the effect of such inhibitors by the addition of pepsin, the reduction being assumed to be due to the capacity of pepsin to degrade certain proteinaceous materials such as edestin and globulin; however, this is only successful at a pH in the range of 4.0 to 7.0, i.e. at a pH which cannot be maintained for all detergent compositions, especially not for detergent compositions containing alkaline builders.

The inhibitors contained in proteinases are compounds of the polypeptide type, i.e. of such compounds which have a molecular weight in the range of 6000 to 12,000 and 20,000 to 60,000. The removal of these inhibitors is very difficult since high-molecular inhibitors are not dialysable and low-molecular inhibitors (contrary to the high-molecular inhibitors) are thermostable and stable towards acids. In addition, numerous soil types of ordinary soiled household textiles act themselves as inhibitor for the enzymes. Thus, e.g. wheat flour has a substantial inhibiting capacity for trypsin and vegetable proteinases so that detergent compositions containing common trypsin are not effective in removing stains such as sauce and pudding stains.

The enzymes of bacterial origin have the further drawback that bacterial proteinases cleave only certain peptide, bonds, whereas pancreas proteinases of the trypsin type effect cleavage of peptide bonds of non-aromatic amino acids and those of the chymotrypsin cleave the bonds of aromatic amino acids.

Moreover, the specific efiiciency of bacterial enzymes is influenced by the culture medium and the mother cultures used for the preparation of these enzymes so that the substrate specificity is changed from generation to generation and it is hardly possible to prepare enzyme products of constant quality.

It is the object of the present invention to provide a method for preparing water-soluble and inhibitor-free enzymes which are especially suitable for the use in detergent compositions and are able to hydrolyze all proteinaceous matter which may be present as soil on textiles also in the presence of other organic substances. It is another object of the present invention to propose new highly effective detergent compositions which do not show the drawbacks of the known detergent compositions containing enzymes. A still further object of the invention is to provide a process for making such detergent compositions which allows to incorporate the enzymes in the detergent composition without loss of activity.

Thus not only themethod for preparing the suitable enzymes is essential for attaining these objects, but it is also necessary to use certain classes of tensides to achieve the desired effect. In addition, it is advantageous to incorporate certain additives in the detergent compositions to improve the activity and especially the shelf life of the enzyme containing detergent compositions. Further, care must be taken that the other additives normally used in detergent compositions e.g. brighteners and perfumes have no detrimental etfect on the enzymatic activity.

According to the invention, these objects are attained by a method for preparing inhibitor-free, easily watersoluble enzymes being especially suitable for the use in detergent compositions, which method comprises comminuting animal pancreas, in particular pig pancreas, optionally together with other intestinal materials, which have been frozen immediately after slaughtering, extracting the material with an alcohol containing 1-2 carbon atoms, e.g. methyl or ethyl alcohol, treating the extract with a water-insoluble liquid solvent, e.g. butyl alcohol, such as n-butyl alcohol, or ethers, such as diethyl ether, precipitating the enzyme from the resulting clear layer with a water-soluble solvent which is an alcohol containing l-2 carbon atoms, such as methyl or ethyl alcohol, or a water-soluble ketone, e.g. acetone, at temperatures below C. and recovering the enzyme.

The extraction may be carried out with the 1-2 carbon alcohol per se or with the aqueous alcohol. The watersoluble solvent employed to precipitate the enzyme may be employed subsequent to or concurrently with the treatment with the water-insoluble solvent. The water-soluble precipitating solvent may be employed per se or, when employed after the water-insoluble solvent treatment, in aqueous solution.

The enzyme obtained this way and consisting substantially of trypsin is inhibitor-free, easily water-soluble and especially suitable for use in detergent compositions.

The preferred procedure comprises the following steps: mixing the pancreas material comminuted to a particle size below 4 mm. with a 2,5- to 6-fold amount of a to 35% aqueous ethanol solution, allowing the mixture to stand for 10 to 15 hours at room temperature, passing it through a filter, optionally after addition of a filtration aid, adding to the filtrate 10 to parts by weight of butyl alcohol per 100 parts by weight of filtrate, optionally after addition of further water, allowing the filtrate to stand for 10 to 15 hours, separating the resulting clear, adding about equal parts by volume of ethanol, and recovering by filtration the enzyme precipitated after standing 2 to 8 hours at temperatures between +1 and 10 C. and consisting substantially of trypsin.

In this process the residue of the ethyl alcohol extraction consisting substantially of pancreatin is suitably dried with acetone. The solids thus obtained can also be added in small amounts as an enzymatically active component to detergent compositions.

Further, it is advantageous to mix the enzyme or enzyme mixture thus obtained and consisting substantially of trypsin or/and pancreatin with about equal parts of lactose. The addition of lactose effectively improves the enzymatic capacity and primarily the stability of the enzyme.

Further, it has been found that enzyme preparations being especially suitable for the use in detergent compositions are obtained when the enzyme or enzyme mixture consisting substantially of trypsin or/and pancreatin and the lactose is mixed with the 3- to 4-fold amount of an inorganic salt such as sodium sulfate, sodium-bicarbonate and/or a neutral phosphate.

Finally it has been found that the enzymes or enzyme mixtures prepared according to the above-described method will show their full capacity only when the detergent compositions comprise 5.0 to 20% by weight of nonionic tensides and 95 to 80% by weight of builders. According to the invention, 0.1 to 0.6% by weight of the enzyme or enzyme mixture obtained according to the method of the invention are incorporated in these detergent compositions.

Suitable builders for a pre-washing agent according to the invention comprise 40 to 70 parts by weight of anhydrous sodium sulfate, 10 to parts by weight of tetrasodium pyrophosphate, 10 to 15 parts by weight of sodium perpyrophosphate and 5 to 10 parts by weight of acid sodium pyrophosphate.

Suitable builder mixtures for detergent compositions for the main or clear wash comprise substantially 10 to 20 parts by weight of a neutral and 5 to 10% by weight of an acid sodium pyrophosphate, 10 to 20 parts by weight of sodium polyphosphate, 10 to 20 parts by weight of sodium perpyrophosphate, 10 to 20 parts by weight of sodium bi-carbonate and 30 to 50 parts by weight of sodium sulfate.

It is especially advantageous when the detergent compositions of the invention contain, based on the enzyme or enzyme mixture added, the 5- to 10-fold amount of carboxyrnethyl cellulose which is a known soil suspending agent maintains the activity of the enzyme and/or enzyme mixture in the washing lye and acts as protective colloid.

Suitably the enzymes or enzyme mixtures used in the detergent compositions of the invention comprise 10 to 20 parts by weight of an easily water-soluble trypsin prepared by the method of the invention, preferably in admixture with 50 to 60 parts by weight of lactose, and 5 to 25 parts by weight of pancreatin, 0.05 to 1 part by weight of urease, and 70 to parts by weight of an inorganic salt.

Such enzyme combination prepared according to the method of the invention and consisting substantially of trypsin admixed with lactose and further of urease and/ or pancreatin possesses a sufficient range of activity required for detergent compositions.

The urease used in this detergent composition is preferably an urease which is precipitated with acetone at low temperatures from a dialysed solution of aqueous ethyl alcohol and the extract of a previously deoiled soy bean flour.

The process of the invention for making these detergent compositions comprises first preparing a mixture of the surface active agents and the builders, drying this mixture and then adding the enzyme or enzyme mixture or spraying it onto the dry mixture.

The invention is further illustrated by the following examples:

EXAMPLE 1 For preparing an inhibitor-free, readily water-soluble trypsin, 50 kg. pancreas glands from pigs were frozen at 15 C. immediately after slaughtering. After a freezing time of 24 hours, this material was passed in frozen condition through a comminuting device, e.g. a mincing machine having a perforated disc with holes of 3-5 mm. in diameter. To the comminuted pancreas material placed in a container there were added 50 kg. of ethyl a1- cohol and 150 liters of tap water; the mixture was slowly stirred for 30 minutes avoiding formation of an emulsion and then allowed to stand in the covered container for 12 to 13 hours at room temperature.

Experiments with other solvents such as isopropyl alcohol gave less favorable results since the protein structure of the enzyme is unfavorably changed and the contact points of the enzyme and therewith its activity are reduced.

After an aging time of at least 12 hours, 5 kg. of kieselguhr (diatomaceous earth) are added to the mixture which is slowly stirred whereby the pancreas fibers assume after a short time a rope-like appearance. When this stage is reached, the whole batch is passed through a fine sieve to separate the pancreas fiber material from the turbid pancreas juice. The pancreas fiber material can be further squeezed on a filter press and the resulting press juice may be combined with the turbid pancreas juice. About 130 liters of pancreas juice and, based on the dry substance, 20 kg. of pancreas fiber material are obtained. The pancreas juice is made up to 200 liters of liquid by addition of water and mixed with 15 parts by weight of butyl alcohol per parts by weight of juice; the mixture is thoroughly stirred for about 5 minutes and then allowed to stand for 12 hours at room temperature. Upon standing a clear layer and-a turbid layer containing substantially fiber residues and foreign proteins are formed. The clear layer is carefully removed by suction, mixed with 1.15 liters of ethyl alcohol per liter of clear solution, and then aged for 6 hours at C. during which time precipitation of trypsin inhibitor-free trypsin occurs. After an aging time of about 6 hours, the batch is filtered through a suction filter or Buchner funnel whereby a total of 650 g. (dry basis) purest, readily water-soluble and inhibitor-free trypsin are obtained. An analysis of this substance showed that this trypsin contained about 450 to 500 Wilstatter units per g. substance. From the filtrate consisting substantially of butanol, ethyl alcohol and water, the butyl alcohol and ethyl alcohol may be recovered without ditliculties.

The pancreas fiber material obtained after extraction with ethyl alcohol and squeezing on the filter press is stirred with actone in a weight ratio of 1:5 and allowed to stand for about minutes. After this time, the mixture is passed through a filter or a seive and the residue is airdried and ground. This way, about 6 kg. pure pancreatin are obtained containing per g. of substance 20 units lipase, 40 units trypsin and 60 units chymotrypsin (measured by the Wilstatter method).

EXAMPLE 2 The dry inhibitor-free water-soluble trypsin obtained according to Example 1 was stabilized by mixing it with lactose in a weight ratio of 45:55; this mixture was then mixed with an inorganic salt in a weight ratio of :85. This mixing or blending facilitates handling of the highly concentrated active trypsin which is especially advantageous for a later mixing with other detergent components.

The inorganic salt employed may be an iron-free sodium sulfate, neutral phosphate such as e.g. sodium pyrophosphate, or sodium bicarbonate. The mixture thus obtained can be added to detergent compositions in amounts of 0.1 to 0.5% by weight, the detergent compositions comprising preferably 5 to by weight of nonionic detergents and 95 to 80% by weight of builders.

EXAMPLE 3 An enzyme preparation which is especially suitable for detergent compositions, removing stains on protein basis because of its trypsin content and containing also urease for removing urea-containing strains and lipase for removing stains on oil basis, is prepared in the following manner:

(A) For preparing the urease, soy beans or roughly ground soy beans are ground in a cross beater mill to flour which is deoiled by percolation with about the tenfold amount of trichloroethylene, 10 kg. of this deoiled, airdried and again ground soy flour are stirred with 40 liters of an aqueous 27% ethyl alcohol solution for one hour. After an aging time of 12 hours, the material is filtered, whereby about 17 liters of filtrate are obtained. The residue is again stirred with liters of 27% ethyl alcohol for one hour and then filtered. This second filtrate of 19 liters and the first filtrate of 17 liters are combined and dialysed in portions of 2 liters against tap water in a dialysis tube of about 20 cm. in diameter for 2.5 hours each. The dialysed solution is cooled to 0 C. and precipitated with the same volume of acetone at 5 C. After 2 to 3 hours, the clear liquid is decanted and the precipitate separated in a beaker centrifuge and dry washed with acetone of 5 C.

It is also possible to spray the residual liquid with the precipitate after removal of the clear liquid onto a carrier salt, e.g. onto an iron-free sodium sulfate.

By the above procedure, a total of 350 g. of completely trypsin inhibitor-free urease with about 250 units per g. substance were obtained.

(B) For preparing the suitable mixed enzyme body, 100 g. of the urease thus obtained were mixed with 10,000

g. of pancreatin obtained according to Example 1 from the residue of the ether extraction 5,000 g. of trypsin isolated according to Example 1 and admixed with about equal parts by weight of lactose, and 67,900 g. of a neutral inorganic salt, in the present case with iron-free sodium sulfate. This enzyme body was an especially suitable addifive for detergent compositions and may be added in amounts of 0.1 to 1.0% by weight.

EXAMPLE 4 A pre-washing agent was prepared by mixing 25 kg. of fatty amide polyglycol ether, 75 kg. of alkyl phenol polyglycol ether, 50 kg. of water, and

1.0 kg. of optical brightener with slow stirring to form a slurry; the optical brightener was added at the end of the mixing procedure.

The fatty amide polyglycol ether (Dionil W had a viscosity of 15 to 20 Engler degrees or to cps. at 50 C.; the alkyl phenol polyglycol ether (Marlophen 8/4) had a surface of 36.3-41.5 dyn/cm. at 22 C., l g./liter measured by the bubble pressure method. The optical brightener was a mixture of equal parts by weight of Tinopal CH 3584 and Tinopal RB 8 200." The solution thus prepared was sprayed in a mixing drum onto a mixture of builders having the following composition:

200 kg. of neutral tetrasodium pyrophosphate 50 kg. of acid sodium pyrophosphate 100 kg. of sodium. perpyrophosphate containing 10% 2 2 20 kg. of carboxymethyl cellulose 480 kg. of anhydrous sodium sulfate After the solution of surface active substances was sprayed onto the builders and after the mixture was completely dry, 1 to 3 kg. of the enzyme body prepared according to Example 3 were added.

Perfumes may be added either to the solution or separately at the end of the mixing operation; suitably the perfumes are first bound to a water-soluble ethylene oxide wax. The preferred perfumes are products which contain no terpenes or branched-chain hydrocarbons.

Regarding the phosphates employed, it is advisable to use sprayed phosphates since these have an especially good absorption capacity for the nonionic detergents in the spraying process and result in a dry fiowable powder. It should be understood that also any other common additives for detergent compositions may be added.

The detergent composition is suitably adjusted to a pH not above 8.4, measured in a 1% detergent solution at 60 C.

The detergent composition prepared according to this example is effective in a temperature range from 30 to 65 C., the enzyme activity being independent from the water hardness.

EXAMPLE 5 For making a detergent composition to be used as main or clear washing agent, a mixture was prepared from 100 kg. of alkyl phenol polyglycol ether (Marlophen 820) and 20 kg. of fatty acid polyglycol ester (Marlipal SU) by adding these components with continuous stirring to 100 liters of warm water until complete dissolution was obtained. The resulting solution was sprayed in a mixing drum onto a mixture of builders having the following composition:

100 kg. of neutral tetrasodium pyrophosphate 60 kg. of acid sodium pyrophosphate 100 kg. of tripolyphosphate 100 kg. of sodium perpyrophosphate (containing 10% 100 kg. of sodium hydrogen carbonate 20kg. of carboxymethyl cellulose 400 kg. of sodium sulfate.

The other additives, namely 1 kg. of optical brightener and 300 g. of perfume bound to 2 kg. of water-soluble ethylene oxide wax, were added to the solution of the washing active agents shortly before spraying. The perfume bound to the water-soluble ethylene oxide wax may also be added separately to the batch.

After the mixed product is completely dry, about 1 to 3 kg. of the enzyme body of Example 4 are added, the amount of the enzyme being dependent on the intended use of the detergent composition.

The pH of a 1% detergent solution should not exceed 8, 8 at 60 C. If desired, the pH may be shifted into the acid range by addition of sodium pyrophosphate or towards the neutral range by addition of commercial lactic acid.

Comparative tests Washing tests with detergent compositions prepared according to the invention using a reduced washing time and washing temperatures up to 60 C. resulted in a much better stain removal than so far obtained with known enzyme-containing detergent compositions. It was found that the detergent compositions of the invention have an especially favorable effect on the mechanical properties of the treated fabrics.

In general it has been found that when using the products of the invention a full washing effect is achieved at substantially lower temperatures and with much less washing active substance. Furthermore, a uniform brightening of the textiles is obtained which is independent from the respective textile material such as cell wool, cotton, wool, silk, or polyamide fibers. Moreover, the fabric is much less attacked as evidenced by the improved values for the loss in tear and tensile strength. Also the ash content is much lower which shows that the detergent composition of the invention is more easily rinsed out of the fabric and forms no deposits which are responsible for rendering the fabric yellow.

In contrast to the products obtained by the known methods, the enzymes prepared according to the invention, especially trypsin and urease, are substantially watersoluble which allows a uniform distribution of the same in the detergent composition and in the washing solution.

The enzymes obtained according to the invention are furthermore free of natural enzyme inhibitors which cannot be completely removed by the known methods.

With the method of the invention it is possible to remove the soy bean trypsin inhibitor so that now trypsin can be used together with urease without reduction of the activity of the trypsin.

The influence of natural enzyme inhibitors which may be present in the fabric soil is compensated by the high immunity and activity, respectively, of the enzymes prepared according to the invention.

The bacterial count of the enzymes prepared according to the invention is in the order of about 10 whereas the bacterial count of bacterial proteinases is in the range of 10 to 10 Thus there is no danger that the enzymes prepared according to the invention cause an infection during the washing process.

The following examples show similar methods for preparing stable, inhibitor-free enzymes with improved solubility, whereby the treatment with the water-soluble solvent employed to precipitate the enzyme is made concurrently with the treatment with water-insoluble solvent.

EXAMPLE 6 For preparing a stable, inhibitor-free and water-soluble trypsin, l0 kgs. of comminuted pancreas glands were stirred for a period of 40 minutes with 7.5 liters of methyl alcohol until a homogenous mixture was obtained. About 7.5 liters of water were added to the mixture; the mixture was stirred slowly for a period of about 10 minutes and was then allowed to stand at ambient temperature of 25 C. for a period of about 24 hours. After this extraction period the mixture was passed through a filter whereby 16.5 liters of a clear extract were obtained. The extract was cooled to a temperature of about 0 C. whereafter a mixture of 8 liters of acetone and 8 liters of ethyl ether was added. After a period of 15 hours the supernating liquid was decanted whereafter the remaining syrup-like precipitated enzyme was mixed with about 2 liters of ethyl alcohol. A white milky precipitate was formed which was suction-filtered and washed with 800 ml. of acetone. The washed precipitate was air-dried on a filter at room temperature whereby 680 gms. of pure water-soluble trypsin were obtained. An analysis showed an activity of 440 to 460 Wilstatter-units.

EXAMPLE 7 The method of Example 6 was repeated with the exception that bovine pancreas was used as starting material. About 590 gms. of trypsin with an activity of 220 Wilstatter-units were obtained..

EXAMPLE 8 The method of Example 6 was repeated with the exception that the enzyme was precipitated with a 1:1 mixture of n-butyl alcohol and methyl alcohol. About 540 gms. of trypsin with an activity of 520-530 Wilstiitterunits were obtained.

What we claim is:

1. Method for preparing inhibitor-free readily water soluble enzymes comprising comminuting animal panareas which has been frozen immediately after slaughtering, extracting said pancreas with an alcohol containing 1-2 carbon atoms, treating said extract with a waterinsoluble liquid solvent selected from the group consisting of a butyl alcohol and an ether, precipitating said enzyme from the resulting clear layer with a water-soluble solvent selected from the group consisting of an alco hol containing l-2 carbon atoms and a ketone at a temperature below 10 C. and recovering the enzyme.

2. Method according to claim 1 wherein characterized in that the water-soluble solvent employed to precipitate the enzyme is employed concurrently with the treatment with Water-insoluble solvent.

3. Method according to claim 1 wherein said material is comminuted to a particle size below 4 mm.; said extraction is with a 2.6-to-6 fold amount of 15-35% aqueous ethyl alcohol solution; prior to the treatment with said water-insoluble liquid solvent, the filtrate is obtained by passage through a filter as a juice; said water-insoluble liquid solvent is a butyl alcohol in amount of l020 parts by weight per parts by weight of said juices; said enzyme is precipitated from said clear layer by addition of about an equal part by volume of ethanol; and said enzyme is recovered by filtering the enzyme precipitated after 2 to 8 hours at temperature between i-I-l and 10 (3.; said enzyme consisting essentially of trypsin.

4. Method according to claim 3 wherein it comprises extracting the residue of the ethyl alcohol extraction with acetone and drying the product consisting essentially of pancreatin.

5. Method according to claim 4 wherein it comprises mixing the enzyme consisting essentially of trypsin with about equal parts of lactose.

6. Method according to claim 5 wherein the mixture of trypsin and lactose is mixed with a 3- to 4-fold amount of an inorganic salt selected from the group consisting of sodium sulfate, sodium hydrogen carbonate, a neutral phosphate and mixtures thereof.

'7. Method according to claim 4 wherein -20 parts by weight of the enzyme consisting essentially of trypsin are mixed with -60 parts of weight of lactose and 10 parts by weight of inorganic salt selected from the group consisting of sodium sulfate, sodium hydrogen carbonate, a neutral phosphate and mixtures thereof and said enzyme, lactose and inorganic salt are mixed with 5- 25 parts of weight of pancreatin and 0.05-1 part by weight of urease.

References Cited UNITED STATES PATENTS 2,571,126 10/1951 Frederiksen 19566 3,260,654 7/1966 Toccaceli 195-66 LIONEL M. SHAPIRO, Primary Examiner U.'S. Cl. X.R. 252- 

